JP7822652B2 - Development of novel PDL1 single domain antibodies - Google Patents

Description

TECHNICAL FIELD The present invention belongs to the field of biomedical or biopharmaceutical technology, and specifically relates to single domain antibodies that specifically target PDL1 and uses thereof.

BACKGROUND ART PD-1/PDL1 immunotherapy is one of the most successful tumor immunotherapies, revolutionizing tumor treatment and leading the transformation of cancer treatment. Immune checkpoint inhibitors fully utilize the body's own immune system to fight cancer. They kill cancer cells by blocking the PD-1/PDL1 signaling pathway, and have the potential to treat many types of tumors, substantially improving the survival of patients with advanced tumors and becoming a "miracle drug" for tumor patients.

Currently, several PD-1 antibodies and PD-1 inhibitors are already on the market both domestically and internationally. Similar to PD-1 antibodies, PDL1 blocks immune suppression mediated by the binding of PDL1 to PD-1 on the surface of T cells, thereby activating T cells to recognize and kill tumor cells, thereby suppressing tumor growth. Unlike PD-1 monoclonal antibodies, PD-1 antibodies bind to PDL1 expressed on tumor cells or tumor-infiltrating immune cells, whereas PD-1 monoclonal antibodies bind to PD-1.

However, current PD-1 antibodies have the following limitations: (1) In addition to blocking the PD-1 and PDL1 pathways, PD-1 antibodies may also affect the PD-1 and PD-L2 pathways, leading to side effects such as interstitial pneumonia; (2) some patients are ineffective in PD-1 monoclonal antibody treatment or are resistant to PD-1 drugs, and alternative drugs are needed for these patients; (3) the efficacy rate and safety of PD-1 antibodies still need to be improved.

Therefore, there is a strong need in this field to develop new drugs that inhibit the PD-1/PDL1 signaling pathway, which have better therapeutic effects, a wider range of applications, and better safety.

SUMMARY OF THE INVENTION It is an object of the present invention to provide nanobodies against PDL1 and uses thereof.
In a first aspect of the present invention, there is provided a nanobody against PDL1.
In another preferred embodiment, the nanobody against PDL1 can specifically bind to PDL1.

In another preferred embodiment, the complementarity determining regions (CDRs) of the Nanobody against PDL1 are one or more selected from the following group:
(1) CDR1 represented by SEQ ID NO: 7, CDR2 represented by SEQ ID NO: 8, and CDR3 represented by SEQ ID NO: 9;
(2) CDR1 represented by SEQ ID NO: 10, CDR2 represented by SEQ ID NO: 11, and CDR3 represented by SEQ ID NO: 12;
(3) CDR1 represented by SEQ ID NO: 10, CDR2 represented by SEQ ID NO: 13, and CDR3 represented by SEQ ID NO: 14;
(4) CDR1 represented by SEQ ID NO: 10, CDR2 represented by SEQ ID NO: 13, and CDR3 represented by SEQ ID NO: 15;
(5) CDR1 represented by SEQ ID NO: 7, CDR2 represented by SEQ ID NO: 16, and CDR3 represented by SEQ ID NO: 17;
(6) CDR1 represented by SEQ ID NO: 18, CDR2 represented by SEQ ID NO: 19, and CDR3 represented by SEQ ID NO: 20.

In another preferred example, any one of the above amino acid sequences further includes a derivative sequence that has undergone the addition, deletion, modification, and/or substitution of at least one amino acid (e.g., 1-3, preferably 1-2, more preferably 1) and that maintains the ability to specifically bind to PDL1.

In another preferred embodiment, the derived sequence that has undergone the addition, deletion, modification, and/or substitution of at least one amino acid and that maintains the ability to specifically bind to PDL1 has an amino acid sequence that is at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homologous or identical to the sequence.

In another preferred embodiment, the CDR1, CDR2 and CDR3 are separated by the framework regions FR1, FR2, FR3 and FR4 of the VHH chain.
In another preferred embodiment, the nanobody against PDL1 further comprises a framework region FR.
In another preferred embodiment, the framework region FR is derived from the amino acid sequence shown in SEQ ID NO: 1-6.

In another preferred embodiment, the framework region FR is one or more selected from the group consisting of:
(1) FR1 represented by SEQ ID NO: 21, FR2 represented by SEQ ID NO: 22, FR3 represented by SEQ ID NO: 23, and FR4 represented by SEQ ID NO: 24;
(2) FR1 represented by SEQ ID NO: 25, FR2 represented by SEQ ID NO: 26, FR3 represented by SEQ ID NO: 27, and FR4 represented by SEQ ID NO: 24;
(3) FR1 represented by SEQ ID NO: 28, FR2 represented by SEQ ID NO: 29, FR3 represented by SEQ ID NO: 30, and FR4 represented by SEQ ID NO: 24;
(4) FR1 represented by SEQ ID NO: 31, FR2 represented by SEQ ID NO: 29, FR3 represented by SEQ ID NO: 32, and FR4 represented by SEQ ID NO: 24;
(5) FR1 represented by SEQ ID NO: 33, FR2 represented by SEQ ID NO: 22, FR3 represented by SEQ ID NO: 34, and FR4 represented by SEQ ID NO: 24;
(6) FR1 represented by SEQ ID NO: 35, FR2 represented by SEQ ID NO: 36, FR3 represented by SEQ ID NO: 37, and FR4 represented by SEQ ID NO: 24.

In another preferred embodiment, the amino acid sequence of the VHH chain of the nanobody against PDL1 is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or a combination thereof.

In another preferred embodiment, the nanobody against PDL1 includes a humanized antibody, a camelid-derived antibody, or a chimeric antibody.
In another preferred embodiment, the nanobody against PDL1 is a llama-derived antibody.

In a second aspect of the present invention, there is provided an antibody against PDL1, the antibody comprising one or more VHH chains of a Nanobody against PDL1 according to the first aspect of the present invention.
In another preferred embodiment, the amino acid sequence of the VHH chain of the Nanobody against PDL1 is selected from the group consisting of SEQ ID NO: 1, SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, or a combination thereof.

In another preferred embodiment, the antibody against PDL1 may be a monomeric, bivalent, and/or multivalent antibody.
In another preferred embodiment, the antibody against PDL1 is a bivalent antibody.

A third aspect of the present invention provides a chimeric antigen receptor CAR containing an extracellular domain comprising the anti-PDL1 nanobody described in the first aspect of the present invention or the anti-PDL1 antibody described in the second aspect of the present invention.

In another preferred embodiment, the extracellular domain further comprises a signal peptide.
In another preferred embodiment, the extracellular domain further comprises other exogenous proteins.
In another preferred embodiment, the antibody comprising the extracellular domain has the amino acid sequence shown in SEQ ID NO: 1-6.

In another preferred embodiment, the extracellular domain of the antibody has an amino acid sequence that is ≥85%, preferably ≥90%, more preferably ≥95% identical to SEQ ID NO:7-17, or has 1, 2 or 3 amino acid differences compared to SEQ ID NO:1-6.
In another preferred embodiment, the CAR has a structure shown in formula Ia:
L1-Nb-H-TM-C-CD3ζ (Ia)
(wherein
L is either absent or a signal peptide sequence.
Nb is the specific binding domain.
H is either absent or a hinge region.
TM is the transmembrane domain.
C is the costimulatory signal domain.
CD3ζ is an intracellular signaling sequence derived from CD3ζ (including wild-type or mutant/modified forms thereof).
The "-" is a connecting peptide or peptide bond.

In another preferred embodiment, each L is a signal peptide of a protein selected from the group consisting of CD8, GM-CSF, CD4, CD28, CD137, or a mutant/modified version thereof, or a combination thereof.
In another preferred embodiment, the Nb targets PDL1.
In another preferred embodiment, the Nb is a PDL1 nanobody.

In another preferred embodiment, H is a hinge region of a protein selected from the group consisting of CD8, CD28, CD137, IgG, or a combination thereof.
In another preferred embodiment, the H is a human IgG1 Fc hinge region.

In another preferred embodiment, the TM is a transmembrane domain of a protein selected from the group consisting of CD28, CD3ε, CD45, CD4, CD5, CD8, CD9, CD16, CD22, CD33, CD37, CD64, CD80, CD86, CD134, CD137, CD154, CD278, CD152, CD279, CD233, or mutant/modified versions thereof, or combinations thereof.

In another preferred embodiment, C is a costimulatory domain of a protein selected from the group consisting of OX40, CD2, CD7, CD27, CD28, CD30, CD40, CD70, CD134, 4-1BB (CD137), PD-1, Dap10, LIGHT, NKG2C, B7-H3, ICAM-1, LFA-1 (CD11a/CD18), ICOS (CD278), NKG2D, GITR, OX40L, 2B4, TLR, or mutant/modified forms thereof, or combinations thereof.

In a fourth aspect of the present invention, there is provided a recombinant protein comprising:
(i) a Nanobody against PDL1 according to the first aspect of the invention, or an antibody against PDL1 according to the second aspect of the invention, or a chimeric antigen receptor according to the third aspect of the invention; and (ii) an optional tag sequence to assist in expression and/or purification.
In another preferred embodiment, the tag sequence comprises an Fc tag, an HA tag, a GGGS sequence, a FLAG tag, a Myc tag, a 6His tag, or a combination thereof.

In another preferred embodiment, the recombinant protein specifically binds to PDL1.
In another preferred embodiment, the recombinant protein (or polypeptide) comprises a fusion protein.
In another preferred embodiment, the recombinant protein is a monomer, dimer, or multimer.

In another preferred embodiment, the recombinant protein specifically binds to PDL1.
In another preferred embodiment, the tag sequence is an Fc tag.

A fifth aspect of the present invention provides a polynucleotide encoding a protein selected from the group consisting of a nanobody against PDL1 described in the first aspect of the present invention, an antibody against PDL1 described in the second aspect of the present invention, a chimeric antigen receptor described in the third aspect of the present invention, or a recombinant protein described in the fourth aspect of the present invention.

In another preferred embodiment, the polynucleotide encodes a protein selected from the group consisting of a nanobody against PDL1 described in the first aspect of the present invention or an antibody against PDL1 described in the second aspect of the present invention.

In another preferred embodiment, the present invention relates to a nucleic acid molecule encoding a Nanobody against PDL1 of the present invention. The nucleic acid of the present invention may be RNA, DNA or cDNA.
In a sixth aspect of the invention, there is provided an expression vector comprising a polynucleotide according to the fifth aspect of the invention.

In another preferred embodiment, the expression vector is selected from the group consisting of DNA, RNA, a viral vector, a plasmid, a transposon, another gene transfer system, or a combination thereof. Preferably, the expression vector comprises a viral vector, such as a lentivirus, adenovirus, AAV virus, retrovirus, or a combination thereof.

In another preferred embodiment, the expression vector is selected from the group consisting of pTomo lentiviral vector, plenti, pLVTH, pLJM1, pHCMV, pLBS.CAG, pHR, pLV, and the like.
In another preferred embodiment, the expression vector is a pcDNA3.4-hIgG1-Fc2 vector.
In another preferred embodiment, the expression vector further comprises a component selected from the group consisting of a promoter, a transcription enhancer element WPRE, a long terminal repeat sequence LTR, and the like.

A seventh aspect of the present invention provides a host cell that contains an expression vector according to the sixth aspect of the present invention or has incorporated into its genome a polynucleotide according to the fifth aspect of the present invention.

In another preferred embodiment, the host cell comprises a prokaryotic or eukaryotic cell.
In another preferred embodiment, the host cell is selected from the group consisting of E. coli, yeast cells, and mammalian cells.
In another preferred embodiment, the host cells are 293F cells.

An eighth aspect of the present invention provides an engineered immune cell that contains an expression vector according to the sixth aspect of the invention or has an exogenous polynucleotide according to the fifth aspect of the invention integrated into its chromosome.

In another preferred embodiment, the engineered immune cells are selected from the group consisting of:
(i) chimeric antigen receptor αβ T cells (CAR-T cells);
(ii) chimeric antigen receptor γδ T cells (CAR-T cells);
(iii) chimeric antigen receptor NKT cells (CAR-NKT cells);
(iv) Chimeric antigen receptor NK cells (CAR-NK cells).

In another preferred embodiment, the engineered immune cells comprise autologous or allogeneic αβ T cells, γδ T cells, NK T cells, NK cells, or a combination thereof.
In another preferred embodiment, the engineered immune cells are CAR-T cells.

In a ninth aspect of the present invention, there is provided a method for producing a Nanobody against PDL1, the method comprising the steps of:
(a) obtaining a culture comprising a Nanobody against PDL1 by culturing a host cell according to the seventh aspect of the invention under conditions suitable for the production of a Nanobody;
(b) isolating and/or recovering said Nanobodies against PDL1 from said culture; and (c) optionally purifying and/or modifying the Nanobodies against PDL1 obtained in step (b).

In a tenth aspect of the present invention, there is provided a method of producing an engineered immune cell according to the eighth aspect of the present invention, the method comprising the step of obtaining said engineered immune cell by transducing into said immune cell a polynucleotide according to the fifth aspect of the present invention or an expression vector according to the sixth aspect of the present invention.
In another preferred embodiment, the method further comprises the step of detecting the function and efficacy of the resulting engineered immune cells.

In an eleventh aspect of the present invention, there is provided an immunoconjugate comprising:
(a) a nanobody against PDL1 according to the first aspect of the invention, or an antibody against PDL1 according to the second aspect of the invention, or a recombinant protein according to the fourth aspect of the invention; and (b) a conjugated moiety selected from the group consisting of a detectable marker, a drug, a cytokine, a radionuclide, an enzyme, a gold nanoparticle/nanorod, a magnetic nanoparticle, a viral coat protein or a VLP, or a combination thereof.

In another preferred embodiment, the (a) moiety is a nanobody against PDL1 according to the first aspect of the present invention, or an antibody against PDL1 according to the second aspect of the present invention.
In another preferred embodiment, the (a) moiety and the conjugated moiety are conjugated via a chemical bond or a linker.

In another preferred embodiment, the radionuclide includes:
(i) a diagnostic isotope selected from the group consisting of Tc-99m, Ga-68, F-18, I-123, I-125, I-131, In-111, Ga-67, Cu-64, Zr-89, C-11, Lu-177, Re-188, or a combination thereof; and/or (ii) a therapeutic isotope selected from the group consisting of Lu-177, Y-90, Ac-225, As-211, Bi-212, Bi-213, Cs-137, Cr-51, Co-60, Dy-165, Er-169, Fm-255, Au-198, Ho-166, I-125, I-131, Ir-192, Fe-59, Pb-212, Mo-99, Pd-103, P-32, K-42, Re-186, Re-188, Sm-153, Ra223, Ru-106, Na24, Sr89, Tb-149, Th-227, Xe-133, Yb-169, Yb-177, or a combination thereof.

In another preferred embodiment, the conjugated moiety is a drug or toxin.
In another preferred embodiment, the drug is a drug for targeting a disease with high PDL1 expression.
In another preferred embodiment, the disease with high PDL1 expression is selected from gastric cancer, lung cancer, liver cancer, osteosarcoma, breast cancer, pancreatic cancer, lymphoma, and the like.
In another preferred embodiment, the drug is a cytotoxic drug.

In another preferred embodiment, the cytotoxic drug is selected from the group consisting of antitubulin drugs, DNA minor groove binding agents, DNA replication inhibitors, alkylating agents, antibiotics, antifolates, antimetabolites, chemotherapy sensitizers, topoisomerase inhibitors, vinca alkaloids, or combinations thereof.

Examples of particularly useful cytotoxic drugs include, for example, DNA minor groove binding agents, DNA alkylating agents, and tubulin inhibitors. Typical cytotoxic drugs include, for example, auristatin, camptothecin, duocarmycin, etoposide, maytansine and maytansinoids (e.g., DM1 and DM4), taxane, benzodiazepine, or benzodiazepine-containing drugs. Drugs (e.g., pyrrolo[1,4]benzodiazepines (PBD), indolinobenzodiazepines, and oxazolidinobenzodiazepines), vinca alkaloids, or combinations thereof.

In another preferred embodiment, the toxin is an auristatin (e.g., auristatin E, auristatin F, MMAE, and MMAF), chlortetracycline, maytansinoid, ricin, ricin A-chain, combretastatin, duocarmycin, dolastatin, adriamycin, daunorubicin, taxol, cisplatin, cc1065, ethidium bromide, mitomycin, etoposide, teniposide, Selected from the group consisting of vincristine, vinblastine, colchicine, dihydroxyanthracenedione, actinomycin, diphtheria toxin, Pseudomonas aeruginosa exotoxin (PE) A, PE40, abrin, abrin A chain, modeccin A chain, α-octastaphylococcus, gelonin, mitogellin, restrictocin, phenomycin, enomycin, chrysin, crotin, calicheamicin, soapwort (Sapaonaria officinalis) inhibitor, glucocorticoids, or combinations thereof.

In another preferred embodiment, the conjugated moiety is a detectable marker.
In another preferred embodiment, the conjugated moiety is selected from the group consisting of a fluorescent or luminescent marker, a radioactive marker, an MRI (magnetic resonance imaging) or CT (computed tomography) contrast agent, or an enzyme producing a detectable product, a radionuclide, a biotoxin, a cytokine (such as IL-2), an antibody, an antibody Fc fragment, an antibody scFv fragment, a gold nanoparticle/nanorod, a virus particle, a liposome, a magnetic nanoparticle, a prodrug-activating enzyme (such as DT-diaphorase (DTD) or biphenyl hydrolase-like protein (BPHL)), or any form of nanoparticle.

In another preferred embodiment, the immune complex comprises a multivalent (e.g. bivalent) VHH chain of a Nanobody against PDL1 according to the first aspect of the invention.
In another preferred embodiment, the multivalent amino acid sequence of the immune complex comprises multiple repeats of the same or different VHH chains of a Nanobody against PDL1 according to the first aspect of the present invention.

In a twelfth aspect of the present invention, there is provided a use of an active ingredient selected from the group consisting of a nanobody against PDL1 according to the first aspect of the present invention, or an antibody against PDL1 according to the second aspect of the present invention, or a chimeric antigen receptor according to the third aspect of the present invention, or a recombinant protein according to the fourth aspect of the present invention, or an engineered immune cell according to the eighth aspect of the present invention, or an immune complex according to the eleventh aspect of the present invention, or a combination thereof, for the preparation of:
(a) a drug for preventing and/or treating a disease with high PDL1 expression;
(b) A reagent for detecting diseases with high PDL1 expression.

In another preferred embodiment, the reagent is a diagnostic reagent, and preferably, the diagnostic reagent is a detection sheet or a detection plate.
In another preferred embodiment, the diagnostic reagent is for detecting PDL1 protein or a fragment thereof in a sample.
In another preferred embodiment, the disease with high PDL1 expression is selected from gastric cancer, lung cancer, liver cancer, osteosarcoma, breast cancer, pancreatic cancer, lymphoma, and the like.

In a thirteenth aspect of the present invention, there is provided a method for detecting PDL1 protein or a fragment thereof in a sample in vitro, the method comprising the steps of:
(1) contacting the sample ex vivo with a nanobody against PDL1 according to the first aspect of the present invention, or an antibody against PDL1 according to the second aspect of the present invention, or a chimeric antigen receptor according to the third aspect of the present invention, or a recombinant protein according to the fourth aspect of the present invention, or an engineered immune cell according to the eighth aspect of the present invention, or an immune complex according to the eleventh aspect of the present invention, or a combination thereof;
(2) Detecting whether an antigen-antibody complex is formed, where the formation of the complex indicates the presence of PDL1 protein or a fragment thereof in the sample.
In another preferred embodiment, the detection includes diagnostic or non-diagnostic detection.

In a fourteenth aspect of the present invention, there is provided a pharmaceutical composition comprising:
(i) as an active ingredient, a nanobody against PDL1 according to the first aspect of the invention, or an antibody against PDL1 according to the second aspect of the invention, or a chimeric antigen receptor according to the third aspect of the invention, or a recombinant protein according to the fourth aspect of the invention, or an engineered immune cell according to the eighth aspect of the invention, or an immune complex according to the eleventh aspect of the invention, or a combination thereof; and (ii) a pharmaceutically acceptable carrier, diluent or excipient.

In another preferred embodiment, the dosage form of the drug composition is selected from the group consisting of an injection and a freeze-dried formulation.

In another preferred embodiment, the pharmaceutical composition comprises 0.01 to 99.99% of the nanobody against PDL1 described in the first aspect of the present invention, or the antibody against PDL1 described in the second aspect of the present invention, or the chimeric antigen receptor described in the third aspect of the present invention, or the recombinant protein described in the fourth aspect of the present invention, or the immunoconjugate described in the eleventh aspect of the present invention, or a combination thereof, and 0.01 to 99.99% of a pharmaceutical carrier, the percentages being mass percentages in the pharmaceutical composition.

In another preferred embodiment, the concentration of the engineered immune cells in the active ingredient is 1×10 ³ -1×10 ⁸ cells/mL, preferably 1×10 ⁴ -1×10 ⁷ cells/mL.

In a fifteenth aspect of the present invention, there is provided a kit comprising:
(1) a first container containing a nanobody against PDL1 according to the first aspect of the invention, or an antibody against PDL1 according to the second aspect of the invention, or a chimeric antigen receptor according to the third aspect of the invention, or a recombinant protein according to the fourth aspect of the invention, or an engineered immune cell according to the eighth aspect of the invention, or an immune complex according to the eleventh aspect of the invention, or a combination thereof; and/or (2) a second container containing a secondary antibody against the contents of the first container;
or,
The kit comprises a base sheet (support plate) and a detection plate comprising a measurement bar containing the antibody against PDL1 described in the first aspect of the present invention, or the antibody against PDL1 described in the second aspect of the present invention, or the recombinant protein described in the third aspect of the present invention, or the immune complex described in the eighth aspect of the present invention, or a combination thereof.

In another preferred example, the kit further includes instructions, and the kit is used to non-invasively detect PDL1 expression in a subject, as described in the above specification.

In another preferred embodiment, the detection kit is used to detect diseases with high PDL1 expression.
In another preferred embodiment, the disease with high PDL1 expression is selected from gastric cancer, lung cancer, liver cancer, osteosarcoma, breast cancer, pancreatic cancer, lymphoma, and the like.

In a sixteenth aspect of the present invention, there is provided a method for preventing and/or treating a disease with high PDL1 expression, the method comprising the step of administering to a subject in need thereof a nanobody against PDL1 described in the first aspect of the present invention, or an antibody against PDL1 described in the second aspect of the present invention, or a chimeric antigen receptor described in the third aspect of the present invention, or a recombinant protein described in the fourth aspect of the present invention, or an engineered immune cell described in the eighth aspect of the present invention, or an immune complex described in the eleventh aspect of the present invention, or a pharmaceutical composition described in the fourteenth aspect of the present invention, or a combination thereof.

In another preferred embodiment, the subject comprises a mammal, such as a human.
In another preferred embodiment, the disease with high PDL1 expression is selected from gastric cancer, lung cancer, liver cancer, osteosarcoma, breast cancer, pancreatic cancer, lymphoma, and the like.
In another preferred embodiment, the engineered immune cells or CAR immune cells contained in the pharmaceutical composition are cells derived from the subject (autologous cells).

In another preferred embodiment, the engineered immune cells or CAR immune cells contained in the pharmaceutical composition are cells derived from a healthy individual (allogeneic cells).
In another preferred embodiment, the method may be used in combination with other therapeutic methods.
In another preferred embodiment, the other therapeutic methods include chemotherapy, radiation therapy, targeted therapy, and the like.

In a seventeenth aspect of the present invention, there is provided a diagnostic method for a disease with high PDL1 expression, the method comprising the steps of:
(i) collecting a sample from a subject to be diagnosed, and contacting the sample with a nanobody against PDL1 according to the first aspect of the present invention, or an antibody against PDL1 according to the second aspect of the present invention, or a chimeric antigen receptor according to the third aspect of the present invention, or a recombinant protein according to the fourth aspect of the present invention, or an engineered immune cell according to the eighth aspect of the present invention, or an immune complex according to the eleventh aspect of the present invention, or a combination thereof; and (ii) detecting whether an antigen-antibody complex is formed, wherein the formation of the complex indicates that the subject is a confirmed patient with a disease with high PDL1 expression.

In another preferred embodiment, the sample is a blood sample or a throat swab sample, or a sample from another tissue organ.
In another preferred embodiment, the disease with high PDL1 expression is selected from gastric cancer, lung cancer, liver cancer, osteosarcoma, breast cancer, pancreatic cancer, lymphoma, and the like.

In an eighteenth aspect of the present invention, there is provided a method for producing a recombinant polypeptide, wherein the recombinant polypeptide is an anti-PDL1 nanobody according to the first aspect of the present invention, or an anti-PDL1 antibody according to the second aspect of the present invention, or a chimeric antigen receptor according to the third aspect of the present invention, or a recombinant protein according to the fourth aspect of the present invention, the method comprising the steps of:
(a) culturing a host cell according to the fifth aspect of the invention under conditions suitable for expression; and (b) isolating said recombinant polypeptide from the culture.

DESCRIPTION OF THE DRAWINGS
FIG. 1 shows the results of identification of PDL1-Fc protein by SDS-PAGE. FIG. 2 shows the results of ELISA detection of immune sera.

Figure 3 shows the results of detection of PCR products by agarose gel electrophoresis. Here, Figure 3A shows two PCR bands of approximately 1000 bp and 750 bp obtained in the first PCR. The 750 bp fragment was recovered from the gel and used as a template for the second PCR. Figure 3B shows a band of approximately 450 bp obtained in the second PCR. When identified by agarose gel electrophoresis, it was proven to be a VHH fragment. FIG. 4 shows the results of a sequence alignment of the phage display library.

Figures 5 to 9 show the results of phage ELISA of the products of solid-phase panning screening of the phage display library, where red indicates clones that simultaneously bind to both the PDL1 antigen and Fc, blue indicates clones that bind only to the PDL1 target antigen but not to Fc, and green indicates clones that bind only to the PDL1 target antigen but not to Fc and have relatively strong binding to the PDL1 target antigen. Figures 5 to 9 show the results of phage ELISA of the products of solid-phase panning screening of the phage display library, where red indicates clones that simultaneously bind to both the PDL1 antigen and Fc, blue indicates clones that bind only to the PDL1 target antigen but not to Fc, and green indicates clones that bind only to the PDL1 target antigen but not to Fc and have relatively strong binding to the PDL1 target antigen.

Figures 5 to 9 show the results of phage ELISA of the products of solid-phase panning screening of the phage display library, where red indicates clones that simultaneously bind to both the PDL1 antigen and Fc, blue indicates clones that bind only to the PDL1 target antigen but not to Fc, and green indicates clones that bind only to the PDL1 target antigen but not to Fc and have relatively strong binding to the PDL1 target antigen. Figures 5 to 9 show the results of phage ELISA of the products of solid-phase panning screening of the phage display library, where red indicates clones that simultaneously bind to both the PDL1 antigen and Fc, blue indicates clones that bind only to the PDL1 target antigen but not to Fc, and green indicates clones that bind only to the PDL1 target antigen but not to Fc and have relatively strong binding to the PDL1 target antigen. Figures 5 to 9 show the results of phage ELISA of the products of solid-phase panning screening of the phage display library, where red indicates clones that simultaneously bind to both the PDL1 antigen and Fc, blue indicates clones that bind only to the PDL1 target antigen but not to Fc, and green indicates clones that bind only to the PDL1 target antigen but not to Fc and have relatively strong binding to the PDL1 target antigen.

10 and 11 show the results of flow cytometry analysis of the overlap product detected by transient transfection. 10 and 11 show the results of flow cytometry analysis of the overlap product detected by transient transfection.

12 to 14 show the binding of purified antibodies to the recombinant cell line CHO-K1/PDL1 overexpressing PDL1, as detected by flow cytometry. 12 to 14 show the binding of purified antibodies to the recombinant cell line CHO-K1/PDL1 overexpressing PDL1, as detected by flow cytometry. 12 to 14 show the binding of purified antibodies to the recombinant cell line CHO-K1/PDL1 overexpressing PDL1, as detected by flow cytometry.

FIG. 15 shows the results of detecting the PD-1-PDL1 blocking activity of monovalent antibodies. FIG. 16 shows the results of detecting the PD-1-PDL1 blocking activity of bivalent antibodies.

Specific Embodiments The present inventors, after extensive and in-depth research and extensive screening, have for the first time developed several nanobodies against PDL1 and bivalent antibodies constructed therefrom. Specifically, in the present invention, llamas were immunized with human-derived PDL1 protein to obtain a high-quality immune nanobody gene library. PDL1 protein molecules were then coupled to a microplate, and utilizing the principle of antigen-antibody specific binding, an immune nanobody gene library (llama heavy chain antibody phage display gene library) was screened with this form of antigen using phage display technology to obtain PDL1-specific nanobody genes. Furthermore, related experimental results have shown that the PDL1 nanobodies obtained in the present invention and bivalent antibodies constructed therefrom can effectively bind to PDL1 protein and simultaneously possess blocking functions, blocking its interaction with its ligand, PD-1. Based on this, the present invention was completed. The PDL1-targeting nanobodies developed in the present invention are useful as a new therapeutic approach for the targeted treatment of diseases such as gastric cancer, lung cancer, liver cancer, osteosarcoma, breast cancer, pancreatic cancer, and lymphoma.

Terminology As used herein, the terms "antibody of the present invention,""antibody of the present invention,""nanobody against PDL1 of the present invention,""nanobody against PDL1 of the present invention,""nanobody against PDL1," and "nanobody against PDL1" have the same meaning and can be used interchangeably, and all refer to antibodies that specifically recognize and bind to PDL1 protein (including human PDL1 protein).

The antibody numbers and corresponding SEQ ID NOs of the Nanobodies of the present invention are shown in Table 1 below.
Note: Each number in the table represents a sequence number, i.e., "1" represents "SEQ ID NO: 1," and the sequence numbers of CDR1, CDR2, CDR3, FR1, FR2, FR3, and FR4 shown in the table are the numbers of the amino acid sequences.

As used herein, the term "antibody" or "immunoglobulin" refers to a heterotetrameric glycoprotein of approximately 150,000 daltons with similar structural characteristics, consisting of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by a single covalent disulfide bond, while the number of disulfide bonds between heavy chains depends on the immunoglobulin isotype. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable region (VH) at one end followed by multiple constant regions. Each light chain has a variable region (VL) at one end followed by a constant region at the other end. The light chain constant region faces the first constant region of the heavy chain, and the light chain variable region faces the variable region of the heavy chain. Special amino acid residues form an interface between the light and heavy chain variable regions.

As used herein, the terms "single domain antibody," "VHH," "nanobody," and "single domain antibody (adAb, or nanobody)" have the same meaning and can be used interchangeably. They refer to the variable region of the heavy chain of a cloned antibody. A single domain antibody (VHH) consisting of only one heavy chain variable region is constructed and is the smallest fully functional antigen-binding fragment. Typically, an antibody lacking the natural light chain and heavy chain constant region 1 (CH1) is first obtained, and then the heavy chain variable region of the antibody is further cloned to construct a single domain antibody (VHH) consisting of only one heavy chain variable region.

As used herein, the term "variable" refers to the fact that certain portions of the variable regions of antibodies differ in sequence, thereby conferring the binding and specificity of each particular antibody for its particular antigen. However, variability is not uniformly distributed throughout the variable regions of antibodies. It is concentrated in three segments called complementarity-determining regions (CDRs) or hypervariable regions in the light and heavy chain variable regions. The relatively conserved portions of the variable regions are called framework regions (FRs). Naturally occurring heavy and light chain variable regions each contain four FR regions that are primarily in a beta-sheet structure and connected by three CDRs that form connecting loops, sometimes in a partial beta-sheet structure. The CDRs in each chain are closely spaced by the FR regions and, together with the CDRs of the other chain, form the antigen-binding site of the antibody (see Kabat et al., NIH Publ. No. 91-3242, Vol. I, pp. 647-669 (1991)). The constant region is not directly involved in binding between the antibody and the antigen, but exhibits different effector functions, such as participating in antibody-dependent cellular cytotoxicity of the antibody.

As known to those skilled in the art, immunoconjugates and fusion expression products include conjugates of the antibodies of the present invention or fragments thereof conjugated to drugs, toxins, cytokines, radionuclides, enzymes, and other diagnostic or therapeutic molecules. Furthermore, the present invention includes cell surface markers or antigens conjugated to the nanoantibodies or fragments thereof against PDL1.

As used herein, the terms "heavy chain variable region" and "VH" are used interchangeably.
As used herein, the terms "variable region" and "complementarity determining region (CDR)" can be used interchangeably.

In one preferred embodiment of the invention, the heavy chain variable region of the antibody comprises three complementarity determining regions: CDR1, CDR2, and CDR3.
In one preferred embodiment of the invention, the heavy chain of the antibody comprises the heavy chain variable region and heavy chain constant region described above.

In the present invention, the terms "antibody of the present invention," "protein of the present invention," and "polypeptide of the present invention" can be used interchangeably and all refer to a polypeptide that specifically binds to PDL1 protein, such as a protein or polypeptide having a heavy chain variable region. These may or may not contain an initial methionine.

The present invention also provides other proteins or fusion expression products of the antibodies of the present invention. Specifically, the present invention includes any protein or protein complex and fusion expression product (i.e., immune complex and fusion expression product) having a heavy chain containing this variable region, so long as the variable region is similar to or has at least 90%, preferably at least 95%, homology with the heavy chain variable region of the antibody of the present invention.

Generally, the antigen-binding properties of an antibody are characterized by three specific regions located in the heavy chain variable region, called the CDRs (CDRs), which are divided into four framework regions (FRs). The amino acid sequences of the four FRs are relatively conserved and are not directly involved in binding reactions. These CDRs form a ring structure, held together spatially by a beta sheet formed by the FRs. The CDRs in the heavy chain and the corresponding CDRs in the light chain constitute the antigen-binding site of the antibody. Comparison of the amino acid sequences of similar antibodies can determine which amino acids constitute the FR or CDR regions.

The heavy chain variable region of the antibody of the present invention is of particular interest because at least a portion of it is involved in antigen binding. Therefore, molecules having a heavy chain variable region chain of a CDR-containing monoclonal antibody are included in the present invention as long as their CDRs share 90% or more (preferably 95% or more, and most preferably 98% or more) homology with the CDRs identified herein.

The present invention includes not only complete antibodies, but also immunologically active antibody fragments and fusion proteins consisting of antibodies and other sequences. Therefore, the present invention also includes fragments, derivatives, and analogs of the above antibodies.

As used herein, the terms "fragment," "derivative," and "analog" refer to polypeptides that maintain essentially the same biological function or activity as an antibody of the present invention. Fragments, derivatives, and analogs of the polypeptides of the present invention may be (i) polypeptides in which one or more conservative or non-conservative amino acid residues (preferably conservative amino acid residues) have been substituted, whether or not such substituted amino acid residues are encoded by the genetic code; (ii) polypeptides in which one or more amino acid residues have been substituted; (iii) polypeptides in which the mature polypeptide has been fused to another compound (e.g., a compound that extends the half-life of the polypeptide, such as polyethylene glycol); or (iiiV) polypeptides in which an additional amino acid sequence has been fused to the polypeptide (e.g., a leader sequence, a secretory sequence, a sequence for purifying the polypeptide, a protein precursor sequence, or a fusion protein formed with a 6His tag). Based on the disclosure herein, these fragments, derivatives, and analogs are within the scope known to those of skill in the art.

The antibody of the present invention refers to a polypeptide comprising the above-described CDR region that has binding activity to the PDL1 protein. The term also encompasses mutated forms of the polypeptide comprising the above-described CDR region that have the same function as the antibody of the present invention. These mutations include, but are not limited to, the deletion, insertion, and/or substitution of one or more amino acids (usually 1-50, preferably 1-30, more preferably 1-20, and most preferably 1-10) and the addition of one or more amino acids (usually 20 or less, preferably 10 or less, and more preferably 5 or less) to the C-terminus and/or N-terminus. For example, substitution with amino acids with close or similar functions is generally not expected to alter the function of the protein. Furthermore, the addition of one or more amino acids to the C-terminus and/or N-terminus also generally does not alter the function of the protein. The term also encompasses active fragments and active derivatives of the antibody of the present invention.

Variants of the polypeptide include homologous sequences, conservative variants, allelic variants, naturally occurring mutants, induced mutants, proteins encoded by DNA capable of hybridizing with the DNA encoding the antibody of the present invention under high or low stringency conditions, and polypeptides or proteins obtained with antisera against the antibody of the present invention.

The present invention also provides fusion proteins comprising other polypeptides, such as antibodies or fragments thereof. In addition to substantially full-length polypeptides, the present invention also includes fragments of the antibodies of the present invention. Typically, such fragments comprise at least about 50 contiguous amino acids of an antibody of the present invention, preferably at least about 50 contiguous amino acids, more preferably at least about 80 contiguous amino acids, and most preferably at least about 100 contiguous amino acids.

In the present invention, a "conservative variant of the antibody of the present invention" refers to a polypeptide in which, compared to the amino acid sequence of the antibody of the present invention, 10 or fewer, preferably 8 or fewer, more preferably 5 or fewer, and most preferably 3 or fewer amino acids have been substituted with amino acids having similar or close properties. These conservative variant polypeptides are preferably generated by amino acid substitutions as shown in Table 2.

The present invention further provides a polynucleotide molecule encoding the above-mentioned antibody, or a fragment thereof, or a fusion protein thereof. The polynucleotide of the present invention may be in the form of DNA or RNA. DNA forms include cDNA, genomic DNA, or artificially synthesized DNA. The DNA may be single-stranded or double-stranded. The DNA may be the coding strand or the non-coding strand.

Polynucleotides encoding the mature polypeptides of the present invention include coding sequences encoding only the mature polypeptide, coding sequences for the mature polypeptide and various additional coding sequences, and coding sequences for the mature polypeptide (and any additional coding sequences) and non-coding sequences.

The term "polynucleotide encoding a polypeptide" may refer to a polynucleotide that encodes the polypeptide, or may refer to a polynucleotide that further includes additional coding and/or non-coding sequences.

The present invention further relates to polynucleotides that hybridize to the above sequences and have at least 50%, preferably at least 70%, and more preferably at least 80% homology between the two sequences. The present invention particularly relates to polynucleotides that can hybridize to the polynucleotides of the present invention under stringent conditions. In the present invention, "stringent conditions" refers to (1) hybridization and elution at low ionic strength and high temperature, for example, 0.2x SSC, 0.1% SDS, and 60°C, or (2) the use of a denaturing agent during hybridization, for example, 50% (v/v) formamide, 0.1% fetal bovine serum/0.1% Ficoll at 42°C, or (3) hybridization only occurs when the homology between the two sequences is at least 90%, preferably 95%. Furthermore, polypeptides encoded by hybridizable polynucleotides have the same biological functions and activities as the mature polypeptides.

The full-length nucleotide sequence of an antibody of the present invention, or a fragment thereof, can typically be obtained by PCR amplification, recombinant techniques, or artificial synthesis. Synthetic techniques are applicable, particularly when the fragments are short in length, to synthesize the relevant sequence. Typically, multiple small fragments are first synthesized and then concatenated to obtain a longer fragment of the sequence. Alternatively, the coding sequence for the heavy chain can be fused together with an expression tag (e.g., 6His) to form a fusion protein.

Once the relevant sequence is obtained, it can be obtained in large quantities by recombinant techniques. This typically involves cloning the sequence into a vector, introducing it into cells, and then isolating the relevant sequence from host cells grown by conventional methods. Biological molecules (e.g., nucleic acids, proteins) according to the present invention include biological molecules that exist in isolated form.

Currently, it is already possible to obtain the DNA sequence encoding the protein of the present invention (or a fragment or derivative thereof) entirely by chemical synthesis. Furthermore, this DNA sequence may be introduced into various known DNA molecules (or vectors, etc.) or cells well known in the art. Mutations can also be introduced into the protein sequence of the present invention by chemical synthesis.

The present invention further relates to vectors containing the above-described suitable DNA sequences and suitable promoters or control sequences. These vectors can be used to transform suitable host cells so as to express the proteins.

Host cells may be prokaryotic cells, such as bacterial cells, or lower eukaryotic cells, such as yeast cells, or higher eukaryotic cells, such as mammalian cells. Representative examples include bacterial cells such as Escherichia coli, Streptomyces, and Salmonella typhimurium, fungal cells such as yeast, insect cells such as fruit fly S2 or Sf9, and animal cells such as CHO, COS7, and 293 cells.

Transformation of host cells by DNA recombination may be carried out by conventional techniques familiar to those skilled in the art. When the host is a prokaryotic cell, such as E. coli, competent cells capable of absorbing DNA can be collected after the exponential growth phase and treated with the _CaCl2 method, the process of which is well known in the art. Another method uses _MgCl2 . If necessary, transformation may be by electroporation. When the host is a eukaryotic organism, DNA transfection methods such as calcium phosphate precipitation, microinjection, conventional mechanical methods such as electroporation, and lipofection may be used.

The resulting transformant can be cultured using conventional methods to express the polypeptide encoded by the gene of the present invention. Depending on the host cells used, conventional media may be selected for the culture medium. The cells are cultured under conditions suitable for the growth of the host cells. Once the host cells have grown to an appropriate cell density, the selected promoter is induced using an appropriate method (e.g., temperature shift or chemical induction), and the cells are further cultured.

In the above-described methods, the recombinant polypeptide can be expressed intracellularly or at the cell membrane, or can be secreted extracellularly. If necessary, the recombinant protein can be isolated and purified using various isolation methods based on its physical, chemical, and other properties. These methods are well known to those skilled in the art. Examples of these methods include, but are not limited to, conventional renaturation treatments, treatment with protein precipitants (salting out), centrifugation, osmotic shock, sonication, ultracentrifugation, molecular sieve chromatography (gel filtration), adsorption chromatography, ion exchange chromatography, high-performance liquid chromatography (HPLC), and various other liquid chromatography techniques, as well as combinations of these methods.

The antibodies of the present invention may be used alone, or may be conjugated or coupled to a detectable marker (for diagnostic purposes), a therapeutic agent, a PK (protein kinase)-modifying moiety, or any combination thereof.

Detectable markers used for diagnostic purposes include, but are not limited to, fluorescent or luminescent markers, radioactive markers, MRI (magnetic resonance imaging) or CT (computed tomography) contrast agents, or enzymes that produce a detectable product.
Therapeutic agents that can be conjugated or complexed to the antibodies of the present invention include, but are not limited to, 1. radionuclides, 2. biotoxins, 3. cytokines such as IL-2, 4. gold nanoparticles/nanorods, 5. virus particles, 6. liposomes, 7. magnetic nanoparticles, 8. prodrug-activating enzymes (e.g., DT-diaphorase (DTD) or biphenylhydrolase-like protein (BPHL)), and the like.

PDL1 Protein Programmed cell death 1 ligand 1 (PDL1), also known as cluster of differentiation 274 (CD274) or B7 homolog 1 (B7-H1), is a human protein encoded by the CD274 gene. PDL1 is a 40 kDa type I transmembrane protein that has been implicated in suppressing the immune system in certain specific cases (e.g., pregnancy, tissue transplantation, autoimmune diseases, and certain diseases such as hepatitis). Normally, the immune system responds to foreign antigens that accumulate in lymph nodes or the spleen and promotes the development of antigen-specific cytotoxic T cells (proliferation of CD8+ T cells). On the other hand, programmed death receptor 1 (PD-1) can transmit inhibitory signals and reduce the proliferation of CD8+ T cells in lymph nodes when it binds to programmed death ligand 1 (PDL1), and PD-1 can suppress the recruitment of antigen-specific T cells in lymph nodes by regulating the Bcl-2 gene.

PD-1/PDL1
The binding of PDL1 and PD-1 acts as an inhibitory signal that mediates T cell activation, suppresses the killing function of T cells, and plays a negative regulatory role in the body's immune response.

Pharmaceutical Compositions The present invention also provides compositions. Preferably, the composition is a pharmaceutical composition containing the above-described antibody, or an active fragment thereof, or a fusion protein thereof, and a pharmaceutically acceptable carrier. Typically, these substances are formulated in a non-toxic, inert, pharmaceutically acceptable aqueous carrier, and the pH value will vary depending on the properties of the formulated substances and the disease to be treated, but is usually about 5-8, preferably about 6-8. The formulated pharmaceutical composition can be administered by any conventional route, including, but not limited to, intraperitoneal, intravenous, or topical administration.

The pharmaceutical compositions of the present invention contain a safe and effective amount (e.g., 0.001-99 wt%, preferably 0.01-90 wt%, more preferably 0.1-80 wt%) of the above-described antibody (or conjugate thereof) of the present invention and a pharmaceutically acceptable carrier or excipient. Such carriers include, but are not limited to, saline, buffer solution, glucose, water, glycerin, ethanol, and combinations thereof. The pharmaceutical formulation corresponds to the dosage form. The pharmaceutical compositions of the present invention may also be injectable, and can be prepared by conventional methods using, for example, physiological saline or an aqueous solution containing glucose and other excipients. In the case of injections and solutions, the pharmaceutical compositions are prepared under sterile conditions. The dosage of the active ingredient is a therapeutically effective amount, for example, about 10 μg/kg body weight to about 50 mg/kg body weight daily. The polypeptides of the present invention can also be used in combination with other therapeutic agents.

When using the pharmaceutical composition, a safe and effective amount of the immunoconjugate is administered to a mammal. This safe and effective amount is typically at least about 10 μg/kg body weight and often less than about 50 mg/kg body weight, with the preferred dosage being about 10 μg/kg body weight to about 10 mg/kg body weight. Of course, the specific dosage should be determined taking into account factors such as the mode of administration and the patient's health condition, all of which are within the skill of a skilled physician.

Nanobodies Against PDL1 In the present invention, the nanobodies against PDL1 include monomers, dimers (bivalent antibodies), tetramers (tetravalent antibodies), and/or multimers (multivalent antibodies).
In one preferred embodiment of the present invention, the Nanobody against PDL1 comprises a VHH chain having one or more of the amino acid sequences set forth in SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.

In one preferred embodiment of the present invention, the antibody has a detectable marker, more preferably selected from the group consisting of an isotope, a colloidal gold marker, a colored marker, or a fluorescent marker.

Colloidal gold markers may be used by methods known to those skilled in the art. In one preferred embodiment of the present invention, an antibody against PDL1 protein is labeled with colloidal gold to obtain an antibody labeled with colloidal gold.
The nanobody against PDL1 of the present invention can effectively bind to the PDL1 protein.

The present invention also relates to a method for detecting PDL1 protein or a fragment thereof, which basically comprises the steps of obtaining a cell and/or tissue sample, lysing the sample in a medium, and detecting the level of PDL1 protein in the lysed sample.
In the detection method of the present invention, the sample used is not particularly limited, and a typical example is a sample containing cells present in a cell preservation solution.

Kits The present invention also provides kits containing the antibodies (or fragments thereof) or cassettes of the present invention, and in one preferred embodiment of the present invention, the kits further include a container, instructions for use, buffer solutions, etc.

The present invention also provides a detection kit for detecting the level of PDL1 protein, which includes an antibody that recognizes PDL1 protein, a degradation medium for dissolving the sample, and general-purpose reagents and buffers required for detection, such as various buffers, detection markers, and detection substrates. The detection kit may also be an in vitro diagnostic device.

As described above, the antibody of the present invention has broad biological and clinical application value, and its applications cover many fields, including the diagnosis and treatment of diseases associated with PDL1 protein, basic medical research, biological research, etc. One preferred application is for use in clinical diagnosis, prevention, and treatment of PDL1 protein.

The present invention also provides a method for stimulating an immune response by T cells that target mammalian tumor cell groups or tissues, the method comprising administering CAR-T cells of the present invention to a mammal.

In one embodiment, the present invention involves cell therapy in which a patient's autologous T cells (or allogeneic donor cells) are isolated, activated, and genetically modified to generate CAR-T cells, which are then infused back into the patient. This method significantly reduces the incidence of graft-versus-host reactions, and antigens are recognized by T cells without MHC restriction. Furthermore, a single CAR-T can treat all cancers that express that antigen. Unlike antibody therapy, CAR-T cells can replicate in the body, resulting in long-term durability and sustained tumor suppression.

In one embodiment, the CAR-T cells of the present invention can undergo stable in vivo proliferation and persist for several months to several years. Furthermore, the CAR-mediated immune response can be part of an adoptive immunotherapy process, in which the CAR-T cells can induce a specific immune response against tumor cells that highly express the antigen recognized by the CAR antigen-binding domain. For example, the CAR-T cells of the present invention induce a specific immune response against tumor cells that highly express PDL1.

Treatable cancers include non-vascularized or essentially non-vascularized tumors, as well as vascularized tumors. Types of cancer that can be treated with the CAR of the present invention include, but are not limited to, gastric cancer, lung cancer, liver cancer, osteosarcoma, breast cancer, pancreatic cancer, lymphoma, etc.

Generally, as described herein, activated and expanded cells can be used to treat and prevent diseases such as tumors. Therefore, the present invention provides a method for treating cancer, comprising administering a therapeutically effective amount of CAR-T cells of the present invention to a subject in need thereof.

The CAR-T cells of the present invention can be administered alone or in combination with diluents and/or other components, such as IL-2, IL-17, or other cytokines or cell populations, as pharmaceutical compositions. Briefly, pharmaceutical compositions of the present invention comprise the target cell populations described herein, optionally combined with one or more pharmaceutically or physiologically acceptable carriers, diluents, or excipients.

The pharmaceutical composition of the present invention can be administered in a form appropriate for the disease requiring treatment (or prevention). The amount and frequency of administration will depend on factors such as the patient's condition and the type and severity of the patient's disease, or will be determined by clinical trials.

When referring to an "immunologically effective amount,""antitumor effective amount,""tumor suppression effective amount," or "therapeutic amount," the precise amount of the composition of the invention to be administered will be determined by a physician (taking into account individual differences in the patient's (subject's) age, weight, tumor size, extent of infection or metastasis, and disease symptoms). Pharmaceutical compositions containing the T cells described herein can be administered at a dose of ^{10-10 cells} /kg body weight, preferably ^10-10 cells/kg body weight (including all integer values within the range). ^T cell compositions may be administered multiple times at these doses. Cells can be administered by injection techniques known in immunotherapy (e.g., Rosenberg et al., New Eng. J. of Med. 319:1676, 1988). The optimal dosage and treatment plan for a particular patient can be readily determined by one skilled in the medical arts by monitoring the patient's disease status, and treatment can be adjusted accordingly.

Administration of the composition to a subject may be by any convenient means, including aerosolization, injection, oral administration, infusion, implantation, or transplantation. The compositions described herein may be administered to a patient subcutaneously, intradermally, intratumorally, intranodally, intraspinally, intramuscularly, intravenously, or intraperitoneally. In one embodiment, the T cell compositions of the invention are administered to a patient by intradermal or subcutaneous injection. In another embodiment, the T cell compositions of the invention are preferably administered by intravenous injection. The T cell compositions may also be injected directly into a tumor, lymph node, or site of infection.

In one embodiment of the invention, cells activated and expanded by the methods described herein or other methods known in the art for expanding T cells to therapeutic levels are used to administer to a patient in conjunction with (e.g., before, concurrently with, or after) any number of related treatments, including, but not limited to, antiviral therapy, treatment with agents such as cidofovir and interleukin-2, azacitidine (also known as ARA-C), or treatment with natalizumab for MS patients or efalizumab for psoriasis patients, or other treatments for PML patients. In a further embodiment, the T cells of the invention can be used in combination with chemotherapy, radiation, immunosuppressants such as cyclosporine A, azathioprine, methotrexate, mycophenolate mofetil, or FK506, antibodies, or other immunotherapeutic agents. In further embodiments, the cell compositions of the present invention are administered to a patient in combination with (e.g., before, concurrently with, or after) bone marrow transplantation, chemotherapy, such as fludarabine, external beam radiation therapy (XRT), or cyclophosphamide. For example, in one embodiment, a subject may undergo high-dose chemotherapy followed by a peripheral blood stem cell transplant. In some embodiments, after transplant, the subject receives an infusion of expanded immune cells of the present invention. In another embodiment, the expanded cells are administered before or after surgery.

The dosage of the above treatments administered to a patient will vary depending on the precise nature of the condition being treated and the recipient being treated. Dosage rates for administration to humans can be determined according to art-accepted practice. Typically, 1 x ¹⁰ to 1 x ¹⁰ modified T cells of the present invention can be administered to a patient per treatment or course of treatment, for example, by intravenous infusion.

Reagents, materials, and equipment Main reagents: Agar (Sigma, CAT# A1296), peptone (Sigma, CAT# 93926), yeast extract (OXOID, CAT#: LP0021), sodium chloride (Aladdin, CAT#: C111533), potassium chloride (Aladdin, CAT#: P112133), magnesium sulfate (China Pharmaceutical, CAT#: 10013018), magnesium chloride (China Pharmaceutical, CAT#: 10012818), glucose (Seiko, CAT#: GT1991), Sfil (NEB, CAT#: R0123L), T4 DNA ligase (TaKaRa, CAT#: 2011A), PrimeScript ^™ II 1st Strand cDNA synthesis kit (TaKaRa, CAT#: 6210B), NuHi power mix (Shinkai Seibutsu, CAT#: NH9303), 3 M sodium acetate (pH 5.2-6) (Sigma, CAT#: 126-96-5), DNA fragment recovery kit (TakaRa, CAT#: 9761), gel recovery kit (Qiagen, CAT#: 28706), Amane Plasmid Maxi Kit (Amane, CAT#: DP117), HRP-M13 (Sino Biolo, CAT#: 11973-MM05), PE-anti-human IgG (eBioscience, CAT#: 12-4998-82), rabbit anti-llama IgG (H+L) Secondary antibody [HRP] (Novus, CAT# NBP1-75095), SS320 sensitive cells (iCarTab), pComF phage display vector (iCarTab), NHS-biotin (APExBIO, CAT# A8002), HRP-streptavidin (Boster, CAT# BA1088), streptavidin magnetic beads (NEB, CAT# S14205), antibody affinity detection buffer: HBS-EP+ 10X (GE, Cat# BR100669), amino coupling kit (GE, Cat# BR100050), 10 mM glycine 2.5 (GE, Cat#BR100356), S series CM5 chip (GE, Cat#29149603), PBS (Gbico, Cat#14190-250), DMEM (Gbico, Cat#41965-062), RPMI1640 (Gbico, Cat#61870044), FBS (Gbico, Cat#10099-141), genomic DNA purification kit (Lifetech, Cat#K0512), polybrene (Sigma, Cat#107689-10G), LVtransm transfection reagent (iCarTab, Cat#LVTran100), lymphocyte isolation solution (Stem Cell, Cat#18051).

Main materials: 50 mL Falcon centrifuge tubes (Corning, CAT# 352070), electroporation cups (Bio-Rad 0.2 cm), RNase-free 1.5 mL EP tubes (QSP, CAT#: 509-GRD-Q), 200 μL RNase-free PCR tubes (Axygen, PCR-02D-C), T125 shaker flasks (Corning, CAT# 431143), 15 mL Falcon centrifuge tubes (Corning, CAT# 430052), 6-well plates (Corning, CAT# 3516), 96-well plates (Corning, CAT# 3365).

Main equipment: electroporation device (Eppendorf Multiporator), centrifuge (Xiangyi H1650R), constant temperature incubator (Shanghai Jinghong DNP-9052), constant temperature shaking culture incubator (Langyue DZ-85A), clean bench (SUZHOU ANTAI AIR TECH CO., LTD., SW-CJ-1FD), PCR device (Applied Biosystems ABI2720), safety cabinet (Haier, HR40-IIA2), flow cytometer (Thermo Attune Nxt flow cytometer), Thermo 3111 _CO2 incubator, BiaCore T200.

Amino acid sequence SEQ ID NO: 1 (Nanoantibody 17-A06)
MAQVKLEESGGGLVQAGGSLRLSCVASGRSFITYAVGWFRQAPGKEREFVASINWSGAMTYY TDSVNGRFAISRDNAKNTVYLQMNNLKLEDTAVYYCASTISAVTPTNGYQNWGQGTQVTVSS

SEQ ID NO: 2 (Nanobody 17-C12)
MAQVKLEESGGGLVQPGGSLTLSCAASGRTFGFYGWFRQAPGKEREFVAGITWGGSVTSYA DSVKGRFTISRDNAKNTVYLQMNSLKPENTAVYYCARSPRVTTTPREFDVWGQGTQVTVSS

SEQ ID NO: 3 (Nanobody 17-A01)
MAAVQLVDSGGGLVQPGGSLRLSCAASGRTFGFYGWFRQAPGKEREFVAAITWGGSAISYE DSAKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCARSTRVTTNPREYDYWGQGTQVTVSS

SEQ ID NO: 4 (Nanobody 17-G06)
MAAVQLVESGGGLVQPGGSLRLSCAASGRTFGFYGWFRQAPGKEREFVAAITWGGSAISYD DSAKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYCARSMRVTTNPREYDYWGQGTQVTVSS

SEQ ID NO: 5 (Nanobody 23-G3)
MAAVQLVESGGGLVQAGGSLRLSCVASGRSFITYAVGWFRQAPGKEREFVASVNWSGAMTYY ADAVNGRFTISRDNAKNTVYLQMNNLKLEDTAVYYCAATISAVTP TNGYQNWGQGTQVTVSS

SEQ ID NO: 6 (Nanobody 29-H9)
RLQLVESGGGLVQAGGSLRLSCAAASGFTFSTYAMSWYRGVPEKERELVAFISSSDGGGTTYRDSVKGRFTISRDNGKNTVYLQMNSLKPEDTGVYYCARGLAQIWGQGTQVTVSS

SEQ ID NO: 7 (CDR1 of nanobodies 17-A06, 23-G3)
GRSFITYA

SEQ ID NO: 8 (CDR2 of nanobody 17-A06)
INWSGAMT

SEQ ID NO: 9 (CDR3 of nanobody 17-A06)
ASTISAVTPTNGYQN

SEQ ID NO: 10 (CDR1 of nanobodies 17-C12, 17-A01, 17-G06)
GRTFGF

SEQ ID NO: 11 (CDR2 of nanobody 17-C12)
ITWGGSVT

SEQ ID NO: 12 (CDR3 of nanobody 17-C12)
ARSPRVTTTPREFDV

SEQ ID NO: 13 (CDR2 of nanobodies 17-A01, 17-G06)
ITWGGSAI

SEQ ID NO: 14 (CDR3 of nanobody 17-A01)
ARSTRVTTNPREYDY

SEQ ID NO: 15 (CDR3 of nanobody 17-G06)
ARSMRVTTNPREYDY

SEQ ID NO: 16 (CDR2 of Nanobody 23-G3)
VNWSGAMT

SEQ ID NO: 17 (CDR3 of Nanobody 23-G3)
AATISAVTPTNGYQN

SEQ ID NO: 18 (CDR1 of nanobody 29-H9)
GFTFSTYA

SEQ ID NO: 19 (CDR2 of nanobody 29-H9)
ISSDGGGT

SEQ ID NO: 20 (CDR3 of nanobody 29-H9)
ARGLAQI

SEQ ID NO: 21 (FR1 of nanobody 17-A06)
MAQVKLEESGGGLVQAGGSLRLSCVAS

SEQ ID NO: 22 (FR2 of nanoantibodies 17-A06, 23-G3)
VGWFRQAPGKEREFVAS

SEQ ID NO: 23 (FR3 of nanobody 17-A06)
YYTDSVNGRFAISRDNAKNTVYLQMNNLKLEDTAVYYC

SEQ ID NO: 24 (FR4 of nanoantibodies 17-A06, 17-C12, 17-A01, 17-G06, 23-G3, 29-H9)
WGQGTQVTVSS

SEQ ID NO: 25 (FR1 of nanobody 17-C12)
MAQVKLEESGGGLVQPGGSLTLSCAAS

SEQ ID NO: 26 (FR2 of nanobody 17-C12)
YGWFRQAPGKEREFVAG

SEQ ID NO: 27 (FR3 of nanobody 17-C12)
SYADSVKGRFTISRDNAKNTVYLQMNSLKPENTAVYYC

SEQ ID NO: 28 (FR1 of nanobody 17-A01)
MAAVQLVDSGGGGLVQPGGSLRLSCAAS

SEQ ID NO: 29 (FR2 of nanobodies 17-A01, 17-G06)
YGWFRQAPGKEREFVAA

SEQ ID NO: 30 (FR3 of nanobody 17-A01)
SYEDSAKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC

SEQ ID NO: 31 (FR1 of nanobody 17-G06)
MAAVQLVESGGGLVQPGGSLRLSCAAS

SEQ ID NO: 32 (FR3 of nanobody 17-G06)
SYDDSAKGRFTISRDNAKNTVYLQMNSLKPEDTAVYYC

SEQ ID NO: 33 (FR1 of Nanobody 23-G3)
MAAVQLVESGGGLVQAGGSLRLSCVAS

SEQ ID NO: 34 (FR3 of Nanobody 23-G3)
YYADAVNGRFTISRDNAKNTVYLQMNNLKLEDTAVYYC

SEQ ID NO: 35 (FR1 of nanobody 29-H9)
RLQLVESGGGLVQAGGSLRLSCAAS

SEQ ID NO: 36 (FR2 of nanobody 29-H9)
MSWYRGVPEKERELVAF

SEQ ID NO: 37 (FR3 of nanobody 29-H9)
TYRDSVKGRFTISRDNGKNTVYLQMNSLKPEDTGVYYC

The main advantages of the present invention are as follows:
1. The PDL1 nanobody of the present invention can specifically bind to PDL1 with high affinity.
2. The PDL1 nanobody of the present invention has good PD-1-PDL1 blocking activity and high neutralizing activity against PDL1.
3. In the present invention, both monovalent and bivalent PDL1 nanobodies have been constructed, and both have obvious blocking activity.
4. The PDL1 nanobody of the present invention blocks only the PD-1 and PDL1 pathways, and does not affect the PD-1 and PD-L2 pathways, thereby avoiding side effects such as interstitial pneumonia and improving safety.
5. The PDL1 nanobody of the present invention can more comprehensively activate the immune system to kill tumors, which can obviously benefit patients who are PD-1 ineffective or PD-1 drug-resistant, and can also achieve therapeutic effects, thereby broadening the range of patients it can treat.

6. The PDL1 nanobody of the present invention not only inhibits the PD-1-PDL1 pathway, but also blocks the co-inhibitory function of B7.1 and PDL1, which is beneficial to the overall activation of T cell function and cytokine production, thereby achieving better immune efficacy.
7. The PDL1 nanoantibody of the present invention is cheaper and easier to mass-produce than polyclonal antibodies, reducing production costs and thereby lowering the price of drugs produced therefrom.
8. The PDL1 nanobody of the present invention has a wide range of applications, is stable over a wide temperature range, and can exert its activity even at high temperatures. Unlike conventional antibody fragments, the unfolding of the nanobody at high temperatures has been proven to be completely reversible. Furthermore, the nanobody is stable at extreme pH values and can survive in gastric juices.
9. In terms of structure, nanobodies have a hydrophilic aspect, so compared to conventional antibodies, the PDL1 nanobodies of the present invention do not have the solubility and aggregation problems associated with conventional antibodies.

The present invention will be further described below with reference to specific examples. It should be understood that these examples are used merely to illustrate the present invention and do not limit the scope of the present invention. Experimental methods for which specific conditions are not specified in the following examples are generally carried out under conventional conditions, such as those described in Sambrook et al., "Molecular Cloning: A Laboratory Manual" (New York: Cold Spring Harbor Laboratory Press, 1989), or in accordance with the manufacturer's recommendations. Unless otherwise specified, percentages and parts are by weight.

Example 1. Antigen Production Method 1. The sequence of the PDL1 extracellular domain (19AA-238AA) was prepared by gene synthesis, and an IgG1 Fc tag was added to the C-terminus. The sequence was then subcloned into a eukaryotic expression vector to construct an antigen PDL1-Fc protein expression vector.
2. Plasmid was extracted from the constructed PDL1-Fc protein expression vector, and transiently transfected into 293 cells. The cells were then cultured for 8 days, centrifuged to collect the culture supernatant, which was then filtered through a 0.45 μm filter membrane. The filtrate was transferred to a sterile centrifuge tube and purified using a protein A column to obtain PDL1-Fc protein.
3. The PDL1-Fc protein was identified by SDS-PAGE detection.

As shown in Figure 1, SDS-PAGE detection showed that the actual SDS-PAGE molecular weight of PDL1-Fc was approximately 65 kD, which was consistent with the electrophoretic detection results of the PDL1-Fc product from ACRO. Furthermore, the band purity of the PDL1-Fc of the present invention was high, indicating that the expression and purification were effective.
The purified PDL1-Fc protein is an antigen, also called an immunogen or immunogen.

Example 2. Immunization of llama with antigen One llama was immunized subcutaneously at multiple sites with the PDL1-Fc protein antigen prepared above, and the three immunization series are shown in Table 3.

Example 3 Detection of Immune Titer 1.5 mL of peripheral blood was collected after the third immunization in Example 1, and the centrifuge tube containing the collected blood sample was placed in an incubator at 37°C for 1 hour. The blood sample was then transferred to 4°C and placed overnight.
2. The serum was transferred to a new sterile centrifuge tube and centrifuged at 5000 rpm for 20 minutes. Using a 96-well plate pre-coated with the Fc antigen cleaved from PDL1, anti-camel IgG (H+L) secondary antibody and anti-camel VHH secondary antibody were selected, and an ELISA experiment was performed to detect the immune titer.

As shown in the ELISA detection results in Figure 2, when detected with anti-camel IgG (H+L) secondary antibody, the immune serum was positive and had a higher titer than the non-immune (negative) serum.

The above results showed that the immune serum specifically bound to PDL1, and the OD450 value changed gradually with the dilution gradient of the serum, indicating that the immunization was successful.
100 mL of peripheral blood was collected and prepared for library construction.

Example 4 Separation of PBMCs and Obtainment of VHH Antibody Fragments 1.150 mL of the peripheral blood prepared in Example 1 was collected, and PBMCs were separated using a lymphocyte separation solution.

2. RNA was extracted and reverse transcribed using a PrimeScript ^™ II 1st Strand cDNA Synthesis Kit to produce cDNA.
(1) A reaction mixture Mix 1 shown in Table 4 was prepared in a 200 μL PCR tube.
(2) After incubating at 65°C for 5 minutes, the mixture was quickly cooled on ice.
(3) The reaction mixture shown in Table 5 was prepared in the PCR tube.
(4) After mixing evenly by blowing, the mixture was divided into 80 μL/tube and placed in a PCR machine at 42°C for 1 hour, then heat-inactivated at 70°C for 15 minutes. Finally, the cDNA samples were stored on ice or at -20°C for long periods.

3. Amplification of VHH Fragments (1) A first PCR reaction system (50 μL/tube) shown in Table 6 was prepared.
After preparing the PCR reaction system, the PCR machine was set up using the program in Table 7.
(2) Agarose gel electrophoresis of PCR products:
The PCR product was analyzed by electrophoresis on a 1% agarose gel, and a single strand with a molecular weight of approximately 750 bp was separated. The PCR product was recovered using a gel recovery kit, and the concentration was measured using NanoDrop.
(3) A second PCR reaction system (50 μL/tube) according to Table 8 was prepared.
After preparing the PCR reaction system, the PCR machine was set up using the program in Table 9.
(4) Agarose gel electrophoresis analysis of the second PCR product:
The PCR product was analyzed by electrophoresis on a 1% agarose gel, and a VHH fragment with a molecular weight of approximately 400 bp was isolated. The VHH PCR product was recovered using a gel recovery kit, and the concentration was measured using NanoDrop.

Figure 3 shows the results of agarose gel electrophoresis of the PCR products. Figure 3A shows two PCR bands of approximately 1000 bp and 750 bp obtained in the first PCR. The 750 bp fragment was recovered from the gel and used as a template for the second PCR. Figure 3B shows a band of approximately 450 bp obtained in the second PCR. Identification by agarose gel electrophoresis confirmed that it was a VHH fragment.

The VHH fragments were enzymatically digested with SfiI and subcloned into the phage display vector pComF. The ligated products were then electrotransformed into susceptible E. coli SS320 cells to construct a single-domain antibody phage display library.

Example 5. Construction of a phage display library 1. Construction of a phage display vector:
(1) The pCom F vector and the VHH PCR gel recovered product obtained in Example 4 above were each enzymatically digested with SfiI and incubated at 50°C overnight.
(2) The pCom F vector fragment was separated on a 1% agarose gel, and a 5000 bp vector fragment was excised and collected from the gel. At the same time, the PCR enzyme cleavage product was purified using a DNA fragment collection kit and its concentration was measured using NanoDrop.
(3) The enzyme-cleaved pCom F vector and the VHH fragment were ligated with T4 ligase and incubated at 16°C overnight.

2. Electrotransformation of E. coli with phage ligation products:
(1) An electroporation cup, the ligation product, and electrotransformation-competent cells were prepared and pre-cooled on ice.
(2) The pre-cooled library construction ligation products were placed in electrotransformation-competent cells and placed on ice for 1 min. 70 μL of the DNA/competent cell mixture was placed in each electroporation cup, and the electroporation cups were placed on ice.
(3) Electrotransformation was carried out at 2500 V for 5 ms.
(4) Immediately after the electric shock, the cells were resuspended in SOC medium equilibrated to room temperature and cultured in a shaker at 37°C for 1 hour.

(5) 15 mL of the bacterial solution was taken and used for phage rescue. The remaining 5 mL of the electrotransformation product was added to an equal volume of 50% glycerol, mixed uniformly, and then stored at −80°C.
(6) Separately, 20 μL of the bacterial solution was taken and diluted with 980 μL of 2YT medium, and then 100 μL of the diluted product was taken and diluted a second time with 900 μL of 2YT medium. 50 μL of the resulting solution was uniformly spread on an ampicillin-containing LB plate and cultured overnight at 37°C.
(7) The next day, the plates were removed, the number of clones obtained from each ligation was calculated, and the library volume was calculated.
(8) At the same time, 20 single clones from the plate were inoculated into ampicillin-containing 2YT medium and cultured with shaking at 37°C for approximately 6-8 hours. The bacterial suspension was then sequenced (using the common sequencing primer M13R) to calculate the diversity of the library.

As shown in Figure 4, the sequence alignment results of the phage display library indicated that the blank rate and antibody overlap rate of the phage display library were good, and the capacity of the E. coli library was 6.53E8.

Example 6. Renaturation and rescue of phage display library, and phage precipitation 1. The product after electrotransformation in Example 5 was diluted with 2YT to adjust the OD600 to about 0.2, and ampicillin was added to a final concentration of 100 μg/mL. The mixture was placed in a thermostatic shaker and cultured at 37°C and 225 rpm until the OD600 reached 0.5.
2. M13KO7 was added, and the mixture was shaken evenly, then allowed to stand at 37°C for 30 minutes, and further cultured at 37°C and 225 rpm for 1 hour.
3. M13KO7 volume = 10 x volume x OD600 x 5 x ¹⁰⁸ / M13KO7 titer.
4. The bacterial solution was centrifuged at 6000 rpm for 10 minutes, then resuspended in 2YT-AK medium and cultured overnight at 25°C and 200 rpm.
5. The bacterial solution was centrifuged at 10,000 rpm for 15 minutes.

6. The precipitate was discarded, and the supernatant was transferred to a new centrifuge tube. PEG/NaCl in an amount of 1/5 the volume of the bacterial liquid was added to the tube, mixed uniformly, and then allowed to stand at 4°C for 2 hours.
7. The precipitated phage supernatant was centrifuged at 10,000 rpm at 4°C for 30 minutes, the supernatant was discarded, and the precipitate (phage) in each 50 mL centrifuge tube was resuspended in 1 mL of sterile PBS.
8. The resuspended phage was transferred to a 1.5 mL EP tube, placed in a centrifuge, and centrifuged at 12000 g at 4° C. for 5 minutes.
9. The supernatant was transferred to a new 1.5 mL EP tube, 250 μL of PEG/NaCl was added to the tube, and after mixing uniformly, the tube was left to stand at 4° C. for 10 minutes.
10. The mixture was centrifuged at 12,000 g for 10 minutes, the supernatant was discarded, and the mixture was resuspended in 1 mL of PBS.

11. The mixture was centrifuged at 12,000 g for 5 minutes, the precipitate was discarded, and the supernatant was transferred to a new 1.5 mL EP tube.
12. The mixture was centrifuged at 12,000 g for 5 minutes, and the supernatant was transferred to a new 1.5 mL EP tube to obtain the initial phage library.
13. 10 μL of the precipitate was taken and added to 90 μL of 2YT medium, and diluted ^10-1 to ^10-9 in order. 20 μL of the diluted sample from a three-step gradient of ^10-7 , ^10-8 , and ^10-9 was taken and added to 200 μL of pre-prepared ER2738 with an OD600 of 0.5. After uniform mixing, the mixture was placed in a water bath at 37°C and left to stand for 10 minutes. 108 μL of each was then spread onto an LB-AMP solid plate and left at 37°C overnight. The next day, the colonies were counted to determine the titer.
14. Calculation of titer: Select plates with colony counts between 30 and 300, take the average of the two plates, multiply the colony count by the dilution factor, and then multiply by 100 to obtain the titer.
The phage display library that has undergone renaturation, rescue and counting is ready for subsequent solution panning of the phage display library.

Example 7. Solution panning of phage display libraries Streptavidin magnetic beads were used to bind to pre-biotinylated target antigens, which were then incubated with the phage library to be screened. Non-specifically bound phages were washed away, and recombinant phages bound to the target antigens were eluted with TEA and amplified. After 3-4 rounds of screening, single phages were selected and sequenced.

1. Biotinylated target antigen was diluted with PBS to 50, 10, 5, and 5 μg/ml (the concentration used each time was different), mixed with streptavidin magnetic beads at 4°C, and incubated for 1 hour. Free target antigen was then removed by washing.
2. Approximately 6 x 10 ¹¹ Pfu of library phage or the product of the previous amplification was diluted to 600 μL and placed in an ELISA well pre-coated with Fc. Fc removal was performed three times in total to remove Fc-bound phages.
3. The Fc-removed phage was mixed with streptavidin magnetic beads and incubated at 4°C for 1 hour to remove streptavidin-bound phages.
4. The magnetic beads were discarded, and the phage was diluted to approximately 1 mL with 1% PBSA, mixed with the magnetic beads pre-bound with the target antigen in step 1, and incubated at 4°C for 1 hour.

5. Aspirate the phages and wash the magnetic beads 3-5 times with PBS for 2-3 min each time. Simultaneously, wash a 1.5 mL EP tube that was previously blocked with MPBS with PBS.
6. The phages bound to the magnetic beads were eluted with 600 μL of 1×TEA for 10 min, and the eluted product was transferred to a pre-blocked EP tube and neutralized with 300 μL of Tris-HCl.
7. 10 μL of the output product was added to 90 μL of 2YT medium, diluted 100 times to 10-2, and then diluted 10 times to ^10-2 . 20 μL of the diluted sample from the four-step gradient of ^10-1 , ^10-0 , ^10-1 , and ^10-2 was added to 200 μL of pre-prepared ER2738 with an OD600 of 0.5 (for ^10-1 , 20 μL of the undiluted product was added to ER2738). After mixing uniformly, the mixture was placed in a water bath at 37°C and left to stand for 10 minutes. 108 μL of each mixture was then spread onto an LB-AMP solid plate and left at 37°C overnight. The next day, the colonies were counted to determine the titer.
8. Calculation of titer: Plates with colony counts between 30 and 300 were selected, the average value of the two plates was taken, and the colony count was multiplied by the dilution factor and then by the elution volume.
The constructed phage display library was coated with Fc protein, depleted six times, and then incubated with streptavidin magnetic beads pre-bound with biotin-PDL1 to enrich for phages that specifically bound PDL1. This was repeated four times. The input and output data for each round were collected, and the enrichment factor for each round was calculated.

The calculation results shown in Table 10 indicate that the enrichment factor (input/output) of the phage library was clearly enriched, demonstrating that a phage library that specifically binds to PDL1 was successfully constructed.

Example 8. Cell-based panning of phage display libraries. Using a recombinant cell line overexpressing the target protein, the phage library was sequentially incubated with blank cells and cells overexpressing the target protein. After several washes to remove nonspecifically bound phages, the recombinant phages bound to the cell surface were eluted with glycine or TEA and amplified. After 3-4 rounds of screening, single clones were selected and subjected to ELISA detection.
1. One day before, a 1.5 mL EP tube was blocked with 1% PBSA and left at 4°C overnight.
2. ^1x107 target cells and control cells were each washed three times with PBS, resuspended in 10 mL of 1% PBSA, and blocked at low speed on a destaining shaker at room temperature for 1 hour.
3. Control cells were loaded with 1 x 10 ¹¹ phages and incubated for 1 h, and target cells were subsequently blocked.

4. After incubation, the cells were placed in a centrifuge and centrifuged at 1000 g for 5 minutes. The target cell supernatant (1% PBSA) was discarded, and the control cell supernatant was carefully aspirated and placed in the target cell tube. After resuspending, the cells were placed on a shaker and incubated at low speed for 1 hour.
5. The target cells were placed in a centrifuge and centrifuged at 1000 g for 5 minutes (simultaneously, 1.5 mL EP tubes that had been blocked one day earlier were washed three times with PBS). The target cell supernatant was discarded, and the cells were resuspended in 4 mL of PBS. The cells were then placed in separate blocked 1.5 mL EP tubes, centrifuged with PBS, and washed five times. The cells were then transferred to four other EP tubes, and after five subsequent washes, the cells were transferred to the same EP tubes.
6. The cells were resuspended in 200 μL of PBS, and then 200 μL of 2×TEA was added and rapidly blown up. This was repeated until the solution became non-viscous, and then 200 μL of Tris-HCl was added to neutralize the solution, thereby obtaining the product.
7. Measure the titer of the out-library, as in steps 7 and 8 of the solution panning method in Example 7.

Phage library screening can be performed using either the method in Example 7 or Example 8 depending on the experimental conditions, but in the present invention, the method in Example 7 was preferably used.

Example 9. Phage ELISA
The 1.2YT-Amp medium was divided into 96-well deep well plates, and 500 μL per well was added to each well to pick up single clones from the output plate. The plates were cultured at 37°C and 225 rpm until the OD600 reached 0.5. The final two wells, H11 and H12, contained only medium and no clones, and served as blank controls.
2. At the same time, the antigen was coated onto an ELISA plate in CBS at a concentration of 1 μg/mL, 100 μL/well, and coated at 37°C for 2 hours.
3. Separately, one 96-well deep well plate was taken and divided, and 500 μL of 2YT-A medium was added to each well. Using a multichannel pipette, 10 μL of bacterial solution with an OD600 of 0.5 was drawn up and placed in a new 96-well plate. The plate was cultured overnight at 37°C and 225 rpm, and this was used as the sample bacterial solution for sequencing.
4. M13KO7 was added to a bacterial solution with an OD600 of 0.5, mixed uniformly, and then allowed to stand at 37°C for 15 minutes.
5. M13KO7 volume = 10 x volume x OD600 x 5 x ¹⁰⁸ /M13KO7 titer.

6. After the invasion, the bacterial solution was placed in a shaker and cultured at 37°C and 225 rpm for 45 minutes.
7. The bacterial solution was placed in a centrifuge and centrifuged at 4000 rpm for 10 minutes, the supernatant was discarded, and the cells were resuspended in 2YT-AK medium at 800 μL/well, placed on a shaker, and cultured overnight at 30° C. and 210 rpm.
8. At the same time, the antigen was discarded from the ELISA plate, which was then washed three times with PBST washing solution, and blocked with 3% MPBS at 250 μL/well and left overnight at 4° C. An extra blank plate was then blocked to serve as a blank.
9. The next day, the 96-well deep well plate was placed in a centrifuge and centrifuged at 4000 rpm for 10 minutes. The milk in the ELISA plate was discarded and washed four times with 200 μL of PBST. 50 μL of PBST was added to each well, followed by 50 μL of the phage supernatant after centrifugation. The plate was then incubated at 4°C for 1 hour. The supernatant was discarded and the plate was washed five times with PBST. HRP-anti-M13 secondary antibody was diluted in PBST and incubated at 4°C for 45 minutes at 100 μL per well. The secondary antibody was then washed away and the plate was washed five times with PBST. Color development was performed in TMB at room temperature for 10 minutes, stopped with hydrochloric acid, and the readings were read. Clones with high S/N ratios were selected and used for assay in the stock bacterial suspension.

The second and third output products from Example 7 were selected and subjected to phage ELISA detection. They were coated with streptavidin, biotin-PDL1-Fc, and Fc protein, respectively, and the recombinant phage supernatant was taken and subjected to ELISA detection. The control group was a well plate that was simply blocked.

As shown in Figures 5-9, the ELISA detection results for the second and third output products show that red indicates clones that simultaneously bind to both the PDL1 antigen and Fc, blue indicates clones that bind only to the PDL1 target antigen but not to Fc, and green indicates clones that bind only to the PDL1 target antigen but not to Fc, and that have relatively strong binding to the PDL1 target antigen (i.e., clones to be subjected to sequencing). Clones with an S/N ratio of 4 or higher and that do not bind to Fc were selected and sequenced, and the antibody sequences were analyzed.

Upon analysis, solution panning with streptavidin magnetic beads and biotinylated antigen yielded a total of six different antibody sequences, namely, 17-A06, 17-C12, 17-A01, 17-G06, 23-G3, and 29-H9 of the present invention.

The bacterial suspension of the target clone was taken and amplified by PCR. A signal peptide was added to the N-terminus of the VHH, and IgG1-Fc was added to the C-terminus. The PCR product was transiently transfected into HEK293 cells, and the expressed antibody supernatant was taken and subjected to FACS detection.

Example 10. Amplification, transient transfection, and detection of overlapping PCR products 1. First PCR: Amplification of CMV, VHH, and Fc (1) A PCR reaction system [50 μL/reaction] as shown in Table 11 was prepared (multiple CMV and Fc fragments may be produced) (CMV fragment amplification primers: CMV-F and PCom-R1; VHH fragment amplification primers: PCom-F1 and PCom-R2; Fc fragment amplification primers: PCom-F2 and PGK-R).
Here, the PCR reaction program is as follows:
95°C 10 minutes 95°C 15 seconds 56°C 30 seconds 25 cycles 68°C 60 seconds 68°C 10 minutes (2) 50 μL of the PCR product was taken and added to 1/10 volume of 10x loading buffer, and analyzed by electrophoresis on a 1% agarose gel. The band sizes of CMV and Fc were approximately 750 bp, and the band size of VHH was approximately 560 bp.
(3) The target band was excised from the gel, and the PCR product was purified and its concentration was measured using NanoDrop (if the concentration was too high, it was diluted before the subsequent reaction).

2. Second PCR: Linking CMV, VHH, and Fc by overlap expression PCR (1) A PCR reaction system as shown in Table 12 was prepared.
Here, the PCR reaction program is as follows:
95℃ 10 min 95℃ 15 sec 60℃ 30 sec 15 cycles 68℃ 120 sec

(2) After the PCR reaction program was run, 2 μL each of primers CMV-F and PGK-R was added, and the following PCR reaction program was carried out.
The program for the following PCR reaction is as follows:
20 cycles of 95°C for 15 seconds, 60°C for 30 seconds, 68°C for 120 seconds, and 68°C for 10 minutes. (3) The overlapping PCR product was purified using a Takara DNA fragment recovery kit and the concentration was measured using NanoDrop. At least 10 μg of PCR product was required for subsequent cell transfection verification.
(4) The transfection process is similar to that of eukaryotic expression vectors.

Example 11. Transient Transfection and Expression of Single Domain Antibodies 1. Remove the LVTransm transfection reagent and antibody expression vector pcDNA3.4-hIgG1-Fc2 or overlap PCR product from the refrigerator, thaw them at room temperature, and mix thoroughly and homogenously by pipetting up and down. Remove the PBS buffer and warm it to room temperature. 500 μL of PBS was placed in one well of a 24-well plate, and 4 μg of pcDNA3.4-hIgG1-Fc2 was added. Mix thoroughly and homogenously by pipetting up and down. Then, 12 μL of LVTransm was added. Immediately, mix thoroughly and homogenously by pipetting up and down. The mixture was then left to stand at room temperature for 10 minutes. This mixture is called the DNA/LVTransm complex.

2. 532 μL of the DNA/LVTransm complex was added to 1.5 mL of 293F cells and gently mixed to homogenize. The cells were placed in a 37°C, 5% _CO2 incubator and cultured at 130 RPM for 6-8 hours. After that, 1.5 mL of fresh 293 medium was added, and the cells were returned to the incubator for further culture.
3. After culturing for three consecutive days, the culture supernatant was collected by centrifugation and filtered through a 0.45 μm filter membrane, and the filtrate was transferred to a sterile centrifuge tube for subsequent flow cytometry and ELISA detection.

Example 12. Construction of VHH eukaryotic expression vectors Based on the results of ELISA detection of single phage clones in Example 9, positive clones were selected and sequenced to obtain the VHH antibody sequences. The sequences of the obtained VHH antibodies were analyzed, and each gene was synthesized. The resulting VHH antibody sequences were tandemly linked to human IgG1 Fc and subcloned into the expression vector pcDNA3.4-hIgG1-Fc2. After the vectors were verified to be accurate by sequencing, endotoxin-removed plasmids were prepared using a Qiagen Plasmid Maxi Kit for use.

Example 13. Detection of binding of recombinant antibodies to target proteins by flow cytometry 1. CHO-K1 and CHO-K1-PDL1 cell lines were recovered from liquid nitrogen and the cells were adjusted to the logarithmic growth phase.
2. The two types of cells were each divided into several groups, each containing 5 x 10 ⁵ cells.
3. The expressed antibodies were incubated with target cells CHO-K1 and CHO-K1-PDL1, respectively, and after thorough homogeneous mixing, incubated at room temperature for 1 hour.
4. After centrifugation at 800 xg for 5 minutes at room temperature, the antibody-containing supernatant was discarded and the cells were washed three times with PBS.

5.1 μL of anti-human IgG labeled with PE or Alexa Fluor 488 was added, mixed thoroughly and homogenously, and then incubated at room temperature for 30 minutes in the dark.
6. After centrifugation at 800×g for 5 minutes at room temperature, the supernatant containing the secondary antibody was discarded and the cells were washed three times with PBS.
7. The cells were resuspended in 500 μL of PBS and analyzed by flow cytometry.

A CMV promoter, signal peptide, and human IgG1 Fc tag were added to the N-terminus and C-terminus of the candidate antibody sequence by overlap PCR as described in Example 10. The PCR product was purified and transiently transfected into 293 cells as described in Example 11, and the expressed antibody was detected by flow cytometry.

As shown in Figures 10 and 11, the results of detection by FACS showed that 17-A06, 17-C12, 17-A01, 17-G06, 23-G3, and 29-H9 were able to specifically bind to the CHO-K1/PDL1 recombinant cell line, but did not or barely bound to CHO-K1.
The sequence was synthesized and inserted into the expression vector pcDNA3.4-hIgG1-Fc2 for expression and purification of the candidate antibody.

Example 14. Antibody Expression and Purification 1. The LVTransm transfection reagent and single-chain antibody expression vector were removed from the refrigerator, thawed at room temperature, and then mixed thoroughly and homogenously by pipetting up and down. PBS or HBSS buffer was removed and warmed to room temperature. 2 mL of PBS was placed in one well of a 6-well plate, and 130 μg of pcDNA3.4-hIgG1-Fc2 was added. Mix thoroughly and homogenously by pipetting up and down. 400 μL of LVTransm was then added, and immediately mixed thoroughly and homogenously by pipetting up and down. The mixture was then left to stand at room temperature for 10 minutes.
2. The DNA/LVTransm complex was added to 50 mL of 293F cells and gently mixed to homogenize. The cells were placed in a 37°C, 5% _CO2 incubator and cultured at 130 RPM for 6-8 hours. 50 mL of fresh 293 medium was then added, and the cells were returned to the incubator for further culture.

3. After 7 days of continuous culture, the culture supernatant was collected by centrifugation and filtered through a 0.45 μm filter membrane. The filtrate was transferred to a sterile centrifuge tube and the antibody was purified using a protein A column.
Expression vectors for candidate PDL1 antibodies (i.e., 17-A06, 17-C12, 17-A01, 17-G06, 23-G3, 29-H9) were transiently transfected into 293F cells, transfection supernatants were collected, and the candidate antibodies were purified with protein A.
The candidate antibodies were diluted 5-fold starting from 10 μg/mL in a four-step gradient, and the binding of the purified antibodies to the recombinant cell line CHO-K1/PDL1, which overexpresses PDL1, was detected using a flow cytometer.
The flow cytometry results shown in Figures 12-14 indicate that the binding of the candidate antibodies to the overexpressing cell lines changed with decreasing concentrations of the candidate antibodies, demonstrating that the purified 17-A06, 17-C12, 17-A01, 17-G06, 23-G3, and 29-H9 antibodies can all specifically bind to PDL1-overexpressing cell lines.

Example 15 Detection of PD-1-PDL1 Blocking Activity Jurkat-PD-1-NFAT-Luc reporter gene cell line and ^aAPC CHO-PDL1 cells were regenerated and serially subcultured to logarithmic growth phase, and then seeded in a 96-well plate with 2 x 10 ⁴ effector cells Jurkat-PD-1-NFAT-Luc per well, and the target cells ^aAPC CHO-PDL1 at a 1:1 ratio. Gradient-diluted detection antibody (positive antibody: nivolumab; test antibody) was added to the corresponding wells. The antibody was diluted three-fold, and then serially diluted in nine steps to final concentrations of 30 μg/mL, 10 μg/mL, 3.333 μg/mL, 1.111 μg/mL, 0.3704 μg/mL, 0.1235 μg/mL, 0.04115 μg/mL, 0.01372 μg/mL, and 0.004572 μg/mL, respectively. After 18 hours of co-incubation, 25 μL of One-Glo reagent was added to each well, and luciferase activity in the wells was detected using a Tecan M1000pro microplate reader.

As shown in Figure 15, the positive control nivolumab was able to block the PD-1/PDL1 interaction with an EC50 of 0.3217 μg/mL. Of the test antibodies, the PDL1 single domain antibodies 17-A06, 23-G3, and 29-H9 were all able to block the PD-1/PDL1 interaction, while the remaining three antibodies lacked blocking function. However, the EC50 values of the PDL1 candidate antibodies 17-A06, 23-G3, and 29-H9 were higher than those of the positive control antibody nivolumab. This is presumably because the single domain antibodies are small and the epitope space is limited. Therefore, the three antibodies with blocking function were randomly combined to construct a bivalent antibody and further test for blocking activity.

The three candidate antibodies were randomly combined to construct bivalent antibodies, which were expressed in the following forms: 17-A06-G4S-23-G3, 17-A06-G4S-29-H9, and 23-G3-G4S-29-H9. The antibodies were purified and then tested for PD-1-PDL1 blocking activity in Jurkat-PD-1-NFAT-Luc reporter cell line and ^aAPC CHO-PDL1 cells.

As shown in Figure 16, the positive controls nivolumab (PD-1 antibody, NIV20) and atezolizumab (PDL1 antibody, Atezolizumab) were able to block the PD-1/PDL1 interaction, with comparable EC50 values. All of the tested bivalent PDL1 antibodies were able to block the PD-1/PDL1 interaction, with the blocking ability of the bivalent antibodies consistent with that of the positive controls.

Example 16. Affinity detection of recombinant antibodies A PDL1 candidate antibody (Fc tag) was conjugated to a Protein A probe at 5 μg/mL, and gradient diluted PDL1-His was used as the mobile phase to detect affinity at final concentrations of 2.6 μg/mL, 1.3 μg/mL, 0.65 μg/mL, 0.325 μg/mL, and 0.1625 μg/mL, respectively, using OCTET R2.

Affinity detection was performed on the three bivalent PDL1 antibodies using OCTET R2, and the results of the affinity detection are shown in Table 13.
The detection results showed that all three bivalent antibodies had high affinity, with the highest affinity being 4.198×10 ⁻⁹ M, 2.729×10 ⁻⁹ M, and 1.915×10 ⁻⁹ M, respectively.

All documents related to the present invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, after reading the above content of the present invention, those skilled in the art will be able to make various variations and modifications to the present invention, and it should be understood that equivalent forms of these modifications are within the scope of the claims of the present invention.

Claims (12)

A Nanobody against PDL1, characterized in that the complementarity determining regions (CDRs) of the VHH chain of said Nanobody against PDL1 are one or more selected from the group consisting of:
(1) CDR1 represented by SEQ ID NO: 7, CDR2 represented by SEQ ID NO: 8, and CDR3 represented by SEQ ID NO: 9 ;
(2 ) CDR1 represented by SEQ ID NO: 7, CDR2 represented by SEQ ID NO: 16, and CDR3 represented by SEQ ID NO: 17;
( 3 ) CDR1 represented by SEQ ID NO: 18, CDR2 represented by SEQ ID NO: 19, and CDR3 represented by SEQ ID NO: 20. 2. The Nanobody against PDL1 according to claim 1, wherein the VHH chain of the Nanobody against PDL1 further comprises a framework region FR, wherein the framework region FR is one or more selected from the group consisting of:
(1) FR1 represented by SEQ ID NO: 21, FR2 represented by SEQ ID NO: 22, FR3 represented by SEQ ID NO: 23, and FR4 represented by SEQ ID NO: 24 ;
(2 ) FR1 represented by SEQ ID NO: 33, FR2 represented by SEQ ID NO: 22, FR3 represented by SEQ ID NO: 34, and FR4 represented by SEQ ID NO: 24;
( 3 ) FR1 represented by SEQ ID NO: 35, FR2 represented by SEQ ID NO: 36, FR3 represented by SEQ ID NO: 37, and FR4 represented by SEQ ID NO: 24. The nanobody against PDL1 according to claim 1, wherein the amino acid sequence of the VHH chain of the nanobody against PDL1 is selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO: 5, SEQ ID NO: 6, or a combination thereof. A nanobody against PDL1, comprising one or more VHH chains of the nanobody against PDL1 according to claim 1 , characterized in that it is a monomeric, bivalent or multivalent antibody . A polynucleotide encoding a protein selected from the group consisting of the nanobody against PDL1 according to claim 1 or the nanobody against PDL1 according to claim 4. An expression vector comprising the polynucleotide of claim 5 . A host cell comprising the expression vector according to claim 6 . 1. A method for producing a nanobody against PDL1, comprising the steps of:
(a) obtaining a culture comprising a Nanobody against PDL1 by culturing the host cell of claim 7 under conditions suitable for the production of the Nanobody;
(b) isolating and/or recovering said Nanobodies against PDL1 from said culture; and (c) optionally purifying and/or modifying the Nanobodies against PDL1 obtained in step (b). An immunoconjugate characterized in that it comprises:
(a) a nanobody against PDL1 according to claim 1, or a nanobody against PDL1 according to claim 4; and (b) a conjugated moiety selected from the group consisting of a detectable marker, a drug, a cytokine, a radionuclide, an enzyme, a gold nanoparticle/nanorod, a magnetic nanoparticle, a viral coat protein or a VLP, or a combination thereof. The host cell according to claim 7, wherein the host cell is a chimeric antigen receptor ( CAR ) -T cell or a CAR-NK cell, the chimeric antigen receptor (CAR) comprising an extracellular domain, and the extracellular domain comprises the nanobody against PDL1 according to claim 1. 1. Use of an active ingredient, wherein the active ingredient is selected from the group consisting of the nanoantibody against PDL1 according to claim 1 or the nanoantibody against PDL1 according to claim 4, or a combination thereof, and the active ingredient is one of the following:
(a) a drug for preventing and/or treating a disease with high PDL1 expression;
(b) The use as described above, characterized in that it is used for producing a reagent for detecting a disease in which PDL1 is highly expressed. below:
1. A pharmaceutical composition comprising: (a) as an active ingredient, a nanobody against PDL1 according to claim 1, or a nanobody against PDL1 according to claim 4, or a combination thereof; and (ii) a pharmaceutically acceptable carrier , diluent or excipient.